The human eye is designed so that light traverses the anterior portions (cornea, aqueous humor and lens), the vitreous and the anterior layers of the retina before reaching the outer segments of light-sensitive cells or photoreceptors (PROS), i.e., the rods and cones. FIG. 1A provides a general schematic of the retinal components.
Light not absorbed by the visual pigments in PROS (e.g., rhodopsin in rods, various opsins in cones) is absorbed by the adjacent retinal pigment epithelial cells (RPE), which is highly pigmented, and therefore, functions as a dark chamber. Located at the interphase between photoreceptor (PR) and the choroidal blood vessels, the RPE performs key support functions for PR. These include: (i) providing key blood nutrients through various RPE transporters (e.g., glucose, aminoacids); (ii) maintaining the ionic composition of the subretinal space (space between RPE and PR) through sophisticated transport mechanisms; (iii) participating in the “visual cycle” (FIG. 1B), by re-isomerizing a key lipid, cis-retinal, which intimately associates with rhodopsin and is the acceptor for photons; and (iv) phagocytosis (i.e., “eating up and digestion of”) PR outer segments, a key aspect of the renewal of the retina. In the visual cycle, light impinges on cis-retinal and converts it into trans-retinal, which is quickly converted into an alcohol derivative (cis-retinol), which is transported to the RPE for regeneration of cis-retinal. All of these RPE functions are essential for normal vision.
Unlike other epithelial cells, which regenerate themselves continuously through cell division, RPE cells do not divide, and therefore, are more susceptible to accumulation of materials as they age. One RPE cell disposes the debris generated by 30-50 adjacent PR cells. This debris is produced by circadian shedding of PR tips, the oldest part of PROS.
The daily and heavy phagocytic activity of RPE cells results in the accumulation of lipofuscin in their digestive system, i.e., the endo-lysosomal apparatus. Lipofuscin accumulation is a universal and phylogenically conserved marker of aging (Sohal, R. S., Age Pigments, Elsevier Science Ltd., © 1981). All metabolically active post-mitotic cells, including cardiac myocytes, neurons, and RPE cells, show age-related accumulation of lipofuscin within their lysosomal system. For most of these cells, the lipofuscin originates primarily from the incomplete degradation of exhausted organelles (autophagy). Depending upon the tissue analyzed, lipofuscin granules normally have 30-70% of protein content (Sohal, Ibid.). In striking contrast, the lipofuscin deposits of aged RPE cells contain almost no protein (<2%); rather, they are constituted of lipidic pigment derivatives of trans-retinal, generated by the visual cycle (Ng, K. P., et al., Mol. Cell. Proteomics, 7(7), pp. 1397-405, 2008). The most abundant and toxic lipidic pigments found are the bisretinoids, primarily A2E, followed by A2E isomers, oxidized derivatives of A2E, A2-dihydropyridine-phosphatidylethanolamine (A2-DHP-PE), and smaller quantities of other Vitamin A conjugates belonging to the all-trans-retinal dimer series (Sparrow, J. R., et al., Exp. Eye Res., 80(5), pp. 595-606, 2005). The non-enzymatic pathway leading to the formation of these bisretinoids has been elucidated. FIG. 1C provides a general overview of the bisretinoid pathways.
A2E-lipofuscin accumulates linearly with age because this material is refractory to lysosomal enzyme degradation. Beyond a certain threshold, A2E-lipofuscin becomes toxic to RPE cells, which eventually results in their malfunction and death. This deterioration process results in the decrease or loss of the ability of RPE cells to support adjacent PR cells. Loss of PR cells resulting from the toxic effects of lipofuscin on RPE cells is considered a central pathogenetic mechanism in genetic and age-related retinal degenerations.
Age-related macular degeneration (AMD) is the most common cause of blindness, affecting 36% of Americans in their eighth decade of life, with a devastating decrease in their quality of life (De, S., et al., J. Gen. Physiol., 120(2): pp. 147-57, 2002). Moreover, clinical evidence shows that photoreceptors overlying bisretinoid-loaded RPE areas (containing mostly A2E) are the most prone to degeneration (Holz, F. G., et al., Invest. Ophthalmol. Vis. Sci., 42(5): p. 1051-6, 2001). Although decades are generally required for the natural accumulation of A2E in RPE, in some human genetic afflictions, like Stargardt Disease (SD) and Best Disease (BD), A2E reaches pathogenic levels typically by about 30 years of age, typically resulting in blindness in the fourth decade of life.
Lipofuscin-containing A2E has been shown harmful to RPE cells (Sparrow, J. R., et al., Adv. Exp. Med. Biol., 703: p. 63-74, 2010; Fernandes, A. F., et al., J. Biol. Chem., 283(30): pp. 20745-53, 2008; Lakkaraju, A., et al., Proc. Natl. Acad. Sci. U.S.A., 104(26): pp. 11026-31, 2007). Three mechanisms have been postulated as contributing to A2E-mediated RPE-toxicity: (1) the A2E's predisposition to oxidation, which can be spontaneous or light-induced and leads to the formation of reactive oxygen species (ROI); (2) A2E's detergent properties, i.e., intercalation of A2E into lysosomal membranes leads to interference of cholesterol extrusion and accumulation of cholesterol, as observed in cholesterol storage diseases such as Niemann Pick; and (3) A2E's tendency to form hydrophobic crystals, which are most likely detected by innate immune receptors and trigger inflammation.
A2E and its derivatives have intrinsic fluorescence and account for most of RPE-lipofuscin (RPE-LF) autofluorescence (Sparrow, J. R., et al., Invest. Ophthalmol. Vis. Sci., 40(12): pp. 2988-95, 1999). The fluorescence emission of A2E is influenced by the polarity of the environment in which the molecule is immersed (Sparrow, et al., 1999, Ibid.; Ragauskaite, L., et al., Photochem. Photobiol., 74(3): p. 483-8, 2001; De, et al., 2002, Ibid.). Thus, in water, A2E maximally emits at 610 nm, but in non-polar solvents, such as n-butyl chloride, A2E typically exhibits a blue-shifted maximum of 585 nm (Sparrow et al., 1999, Ibid.). The emission maxima of A2E inside RPE cells is generally between 565 and 570 nm (Sparrow, et al., 1999, Ibid.; Haralampus-Grynaviski, N. M., et al., Proc. Natl. Acad. Sci. U.S.A., 100(6): p. 3179-84, 2003). This fluorescence spectrum suggests that, when inside a cell, A2E is tightly protected against the solvatochromic shift caused by water molecules (Sparrow, et al., 1999, Ibid.). Moroever, it is known that, with aging, the RPE-LF fluorescence shifts even more toward blue, suggesting that with time A2E deposits may adopt stiffer organization inside these granules (Delori, F. C., et al., Invest. Ophthalmol. Vis. Sci., 36(3): pp. 718-29, 1995).
High-magnification transmission electron-microscopy (TEM) has revealed that A2E deposits are housed within the interior of discrete membrane-bound organelles that are uniformly dense, roughly spherical, and approximately 1 micrometer in diameter (Haralampus-Grynaviski, et al., 2003, Ibid.; Bindewald-Wittich, A., et al., Invest. Ophthalmol. Vis. Sci., 47(10): pp. 4553-7, 2006). Data obtained using atomic force microscopy and purified granules has revealed that the bulk of A2E deposits in RPE cells reside in the lumen of these post-lysosomal bodies, forming an orderly aggregated structure (Ng, et al., 2008, Ibid.; Sparrow, et al., 1999, Ibid.). Because of their ultrastructural appearance, A2E-containing formations are sometimes also referred to as “lipofuscin granules”.
Some studies have predicted that positively-charged amphipathic lipids with cone-shaped molecular geometry, like A2E, can self-assemble in water as an inverted hexagonal phase creating large hydrophobic semi-crystalline constructions (Seong, S. Y., et al., Nat. Rev. Immunol., 4(6): pp. 469-78, 2004; Klymchenko, A. S., et al., Nanoscale, 2(9): pp. 1773-80, 2010). Significantly, highly repetitive hydrophobic structures are known to be among the strongest inducers of chronic inflammation (Seong, et al., 2004, Ibid.). For example, monosodium urate, silica crystals, and asbestos are known to form hydrophobic crystals that are highly pro-inflammatory (Martinon, F., et al., Annu. Rev. Immunol., 27: pp. 229-65 (2009); Martinon, F., et al., Immunol. Rev., 233(1): pp. 218-32, 2010; Martinon, F., Curr. Rheumatol. Rep., 12(2): pp. 135-41 (2010); Dostert, C., et al., 320(5876): pp. 674-7, 2008).
Current therapeutic approaches aimed at alleviating vision loss and retinal (for example, macular) diseases associated with A2E accumulation generally rely on retarding A2E formation by drugs or viral-based gene delivery methods. Current drug therapy generally involves decreasing all-trans-retinal formation, which generally causes sight loss, including night blindness, as a side effect (Zarbin, M. A., et al., Retina, 30(9): pp. 1350-67, 2010). Current gene therapy approaches generally involve delivering the WT gene to individuals with genetic mutations, but this approach is not applicable to AMD. Significantly, neither of these methodologies has been shown to effectively retard or reverse the accumulation of A2E once such accumulation has occurred.